Human parvovirus B19 (sometimes called erythrovirus) is a blood borne, non-enveloped virus that has a single-stranded DNA (ssDNA) genome of about 5.5 kb (Shade et al., 1986, J. Virol. 58(3): 921-936, Brown et al., 1997, Ann. Rev. Med. 48: 59-67). Individual virions contain one copy of either the plus or minus strand of the genome, represented in approximately equal numbers. The ssDNA genome has inverted terminal repeats that form 5′ and 3′ hairpins of about 350 nt which are essential for viral replication. The genome includes two open reading frames on the plus strand which code for structural proteins (VP1 and VP2) and non-structural protein (NS1).
Infection with parvovirus B19 can occur via respiratory transmission or through infected blood or blood products. Viremia can reach high levels (e.g., up to 1011 per ml of blood) at about a week after inoculation, but is generally cleared within about two weeks following infection. Infected individuals may exhibit no symptoms, or have erythema infectiosum symptoms that include mild flu-like symptoms, rash, and/or temporary arthritis-like joint pain (arthropathy). Children are more likely than adults to develop the rash (called “fifth disease”), whereas arthropathy is a common symptom in adults. More serious problems occur in susceptible patients, including aplastic crisis in patients with hemolytic anemias, and persistent parvovirus infection and other hematologic changes in immunosuppressed patients. In women, parvovirus B19 infections have been associated with loss of about 10% of early pregnancies due to fetal death.
Parvovirus B19 is a relatively resistant to viral inactivation, e.g., by chemical or heat-treatment methods used to destroy infective particles in blood, serum or plasma. Also, high viral concentrations in a sample may overwhelm viral depletion methods used to remove viral contaminants from the sample. Parvovirus B19 in blood, plasma or plasma-derived products can infect additional individuals who receive contaminated transfusions or products. Plasma derivatives are often made from pooled donations (e.g., a pool of thousands of individual donations) resulting in the risk that a single contaminated donation could contaminate the pool and products derived from it. Thus, there is a need to detect the presence of human parvovirus B19 in biological samples, such as donated blood or plasma to prevent further infection. There is also a need for an assay that detects parvovirus with a sensitivity that allows detection of low titres of virus as may occur early in an infection or in diluted or pooled samples. An assay for parvovirus B19 nucleic acid which detects an appropriate level of contamination may facilitate removal of infected donated units from the blood supply or contaminated lots of pooled plasma before use.
Immunodiagnostic methods have been used to identify blood, serum or plasma that is potentially contaminated with parvovirus B19. Many methods detect anti-parvovirus antibodies (IgM or IgG) present in an individual's serum or plasma (e.g., see PCT Nos. WO 96/09391 by Wolf et al. and WO 96/27799 by Hedman et al.). Immunological methods, however, have limitations on detecting recent or current infections because they rely on detecting the body's response to the infectious agent. Because of the rapid rise in viremia following infection, an individual's blood may contain high levels of parvovirus B19 before anti-parvovirus antibodies are detectable, leading to false negative results. Because viremia is often quickly cleared, a person may remain antibody-positive even when infective particles are not present, leading to false positive results. Also, up to about 90% of adults are seropositive for parvovirus B19, making accurate immunological detection of recent or current infections difficult. Other assays detect the presence of parvovirus B19 by detecting the virus or empty viral capsid bound to a purified cellular receptor (U.S. Pat. No. 5,449,608 to Young et al.).
DNA hybridization and amplification methods have been used to detect human parvovirus B19. U.S. Pat. No. 5,688,669 to Murtagh et al. describes detection of parvovirus B19 by amplifying a 284 bp portion of parvovirus B19 DNA by using PCR and then digesting the amplified dsDNA with exonuclease to make ssDNA. The ssDNA was hybridized with two separate oligonucleotide probes, a capture probe and a detection probe, and hybrids were detected on a solid support, e.g., by using a colorimetric assay in a microtiter plate. U.S. Pat. No. 6,183,999 to Weimer et al. discloses a nucleic acid amplification assay to detect high-titer parvovirus B19 in plasma protein solutions. By amplifying DNA in the NS1 gene using suboptimal annealing and elongation temperatures, the assay is used to indicate the presence of exceptionally large amounts of pathogenic viruses in the sample. PCT No. WO 96/09391 by Wolf et al. describes cloning of the sequence that encodes the NS-1 protein by linking PCR amplified fragments. Nested PCR and/or dot-blot assays were used to detect parvovirus DNA in patients' sera, and results were correlated with symptoms and a humoral immune response to the NS-1 protein. PCT No. WO 99/28439 by Nguyen et al. discloses the genome sequence of parvovirus B19 and fragments useful as diagnostic and immunogenic agents. PCT No. WO 99/43362 by Barrett et al. discloses a quantitative test based on PCR amplification to demonstrate that plasma proteins were free of human parvovirus B19 following filtration to eliminate the pathogens. PCT No. WO 01/06019 by Lazo et al. discloses DNA oligonucleotide sequences that can hybridize to human and porcine parvovirus sequences. These oligonucleotides can serve as primers in a PCR reaction to amplify portions of the parvovirus DNA in the NS and VP regions, and as probes to detect amplified sequences. The porcine parvovirus is introduced into a sample as an internal control that is co-purified and co-amplified with the human parvovirus B19 DNA. PCT No. WO 01/14593 by Zerlauth et al. discloses a method for detecting contaminating microorganisms, such as parvovirus B19, in pooled biological samples by using two nucleic acid amplification processes that have different predetermined detection sensitivities, which is used to identify and eliminate contaminated samples. The assay first tests a screening pool made up of combined aliquots of multiple samples by using nucleic acid amplification to detect, at a first detection limit, the presence of a microorganism's nucleic acid. Next, a subpool from the positive screening pool is tested by using a second nucleic acid amplification that has a less sensitive detection limit compared to the first detection limit. An example of the method tested plasma by using PCR amplification of a fragment of parvovirus B19 DNA and detection using fluorescently-labeled probes.
PCT No. WO 02/00924 by Tijssen et al. discloses nucleic acid sequences from various parvoviruses, including human parvovirus, that contain sequences coding for viral phospholipase A2 proteins or related polypeptides. These are useful for identifying agents capable of inhibiting viral phospholipase A2 activity or expression, including antisense oligonucleotides, or for making improved recombinant vectors for gene therapy.
JP 04088985 by Sugamura et al. discloses a cloned gene encoding human parvovirus B19 protein VP-1, which was cloned by using PCR to amplify DNA fragments that were integrated into a cloning vector.